Abstract:

Objective To construct a eukaryotic expression vector for transporters associated with antigen processing gene fused with enhanced green fluorescent protein (EGFP) gene and to analyze the expression and subcellular localization of the fusion protein in A375 cells. Methods The monoclone A375 cells with stable expression of TAP were obtained by G418 selection and were identified with checking EGFP expression by FCM and observing under Fluorescence microscope. Each plasmid (pTAP1-EGFP, or pTAP2-EGFP), together with pDsRed2-ER vector, were transiently transfected into A375 cells,and the localizations of expressed proteins were analyzed by confocal microscopy. Results pTAP1-EGFP and pTAP2-EGFP were used to restore the expressions of TAP1 and TAP2 in the antigen presentation pathway-deficient human MM cell line, A375. Transfection of A375 cells with pTAP1-EGFP and pEGFP-TAP1+TAP2 increased the expression of TAP1 and TAP2. The localizations of TAP1-EGFP and TAP2-EGFP overlapped with that of the ER marker, indicating that TAP protein localized subcellularly on the ER. Conclusions The expression vector for TAP-EGFP fusion gene has been constructed successfully and expressed in A375 cells. The expressed fusion protein has similar subcellular localization to pDsRed2-ER. pTAP-EGFP showed efficacy in increasing TAP mRNA and protein in A375 cell lines.